Free polyunsaturated fatty acid-containing composition and manufacturing method therefor

ABSTRACT

The present disclosure is: a free polyunsaturated fatty acid-containing composition, which comprises at least one free polyunsaturated fatty acid having 20 or more carbon atoms, the content being at least 80.0% of the fatty acids in the composition, and satisfies at least one selected from a group consisting of conditions (1) and (2): (1) the content of conjugated unsaturated fatty acid is 1.0% or less of the fatty acids in the composition, and (2) the Gardner color is less than 3+; and a manufacturing method for the free polyunsaturated fatty acid-containing composition comprising the preparation of a raw material composition containing at least one polyunsaturated fatty acid having 20 or more carbon atoms, and hydrolysis of a reaction solution containing the prepared raw material composition, a lower alcohol, water and an alkali catalyst at a temperature of 10° C. or lower.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation application of International Patent ApplicationNo. PCT/JP2016/075444 filed Aug. 31, 2016, which claims the benefit ofJapanese Patent Application No. 2015-170856, the full contents of all ofwhich are hereby incorporated by reference in their entirety.

BACKGROUND Technical Field

The present disclosure relates to a free polyunsaturated fattyacid-containing composition and a manufacturing method therefor.

Description of the Related Art

Long-chain polyunsaturated fatty acids having 20 or more carbons, suchas eicosadienoic acid, dihomo-γ-linolenic acid (DGLA), eicosatetraenoicacid, arachidonic acid (ARA), eicosapentaenoic acid (EPA),docosatetraenoic acid, docosapentaenoic acid, and docosahexaenoic acid(DHA), have been known to exhibit various functionalities in organisms.Therefore, use of polyunsaturated fatty acids as functional componentsin products such as medicaments, health food, and cosmetics has beenstudied. Accordingly, there has been a demand for the production ofpolyunsaturated fatty acids in large quantities at high concentrations.

In many cases in natural, polyunsaturated fatty acids are present inoils as constituent fatty acids of triacylglycerol (glyceride).Therefore, to obtain a free polyunsaturated fatty acid, hydrolysis of aconstituent fatty acid in triacylglycerol or a fatty acid alkyl ester istypically performed.

For example, WO 2013/172346 discloses that a (free) polyunsaturatedfatty acid is obtained by hydrolyzing an ester of polyunsaturated fattyacid obtained by a combination of rectification and columnchromatography.

WO 2015/083843 discloses that a free fatty acid of DGLA is obtained byhydrolyzing a DGLA lower alkyl ester, which is obtained by producing alower alkyl ester of a microbial oil and then rectifying using an alkalicatalyst to enhance purity.

SUMMARY

In hydrolysis treatment to obtain a free fatty acid, a substance that isnot present or that is present in a small amount in the raw materialbefore the treatment may be generated or increased, in addition to thetarget free polyunsaturated fatty acid. Such a substance is an impuritythat is not the target substance, and often has an unidentifiedstructure or function. Therefore, it is desired that the content thereofis made as small as possible in the free polyunsaturated fattyacid-containing composition.

Furthermore, to sufficiently exhibit functions of a free polyunsaturatedfatty acid, a composition containing high concentration of the freepolyunsaturated fatty acid has been desired, and the concentration of afree polyunsaturated fatty acid has been increased by concentrationtreatment or the like. The impurities described above may includeimpurities that are difficult to be removed by following processes dueto similarity in structure with the target free polyunsaturated fattyacid or the like. In this case, it is concerned that the concentrationof impurities may also increase as the target free polyunsaturated fattyacid is concentrated. It is also considered that precision of therefining is enhanced to remove impurities; however, a limit exists forenhancing precision, and it is industrially disadvantageous from theperspective of treatment efficiency, treatment time, and the like.

Therefore, demands exist for free polyunsaturated fatty acidcompositions having less impurities and manufacturing methods for a freepolyunsaturated fatty acid composition having less impurities.

The present disclosure includes the following aspects:

[1] A free polyunsaturated fatty acid-containing composition, containingat least one free polyunsaturated fatty acid having 20 or more carbonsin an amount that a content thereof is 80.0% or greater of fatty acidsin the composition, and the free polyunsaturated fatty acid-containingcomposition satisfies at least one selected from the group consisting ofconditions (1) and (2):

(1) a content of a conjugated unsaturated fatty acid being 1.0% or lessof the fatty acids in the composition; and

(2) the Gardner color being less than 3+.

[2] The free polyunsaturated fatty acid-containing composition accordingto [1], where an anisidine value is 5.0 or less.

[3] The free polyunsaturated fatty acid-containing composition accordingto [1] or [2], where a content of the fatty acid alkyl ester is 0.2% orless of the fatty acids in the composition.

[4] The free polyunsaturated fatty acid-containing composition accordingto any one of [1] to [3], where the content of the conjugatedunsaturated fatty acid is from 0.001% to 1.0% of the fatty acids in thecomposition.

[5] The free polyunsaturated fatty acid-containing composition accordingto any one of [1] to [4], where the polyunsaturated fatty acid is atleast one selected from the group consisting of eicosadienoic acid,dihomo-γ-linolenic acid, Mead acid, eicosatetraenoic acid, arachidonicacid, eicosapentaenoic acid, docosatetraenoic acid, docosapentaenoicacid, and docosahexaenoic acid.

[6] The free polyunsaturated fatty acid-containing composition accordingto any one of [1] to [5], where a total content of a residual organicsolvent in the composition is 5000 ppm or less.

[7] The free polyunsaturated fatty acid-containing composition accordingto any one of [1] to [6], where a content of a di- or higher-valentpolyunsaturated fatty acid having 18 carbons in the composition is 2.0%or less of the fatty acids in the composition.

[8] A manufacturing method of a free polyunsaturated fattyacid-containing composition, the method including:

providing a raw material composition containing at least onepolyunsaturated fatty acid having 20 or more carbons; and

performing hydrolysis treatment on a reaction solution containing theprovided raw material composition, a lower alcohol, water, and an alkalicatalyst in a temperature condition at 10° C. or lower.

[9] The manufacturing method according to [8], where the polyunsaturatedfatty acid in the raw material composition is a polyunsaturated fattyacid alkyl ester.

[10] The manufacturing method according to [8] or [9], the methodfurther including adding an acid in a reaction solution after thehydrolysis treatment to terminate the hydrolysis reaction, a pH of thereaction solution after the acid addition being from pH 1.0 to 6.0.

[11] The manufacturing method according to any one of [8] to [10], wherean amount of the lower alcohol in the reaction solution is from 0.9equivalents to 32.0 equivalents relative to an amount of the fatty acidsin the raw material composition.

[12] The manufacturing method according to any one of [8] to [11l],where an amount of the lower alcohol in the reaction solution is from0.20 to 8.20 in terms of weight content ratio relative to the water.

[13] The manufacturing method according to any one of [8] to [12], wherean amount of the water in the reaction solution is from 6.0 equivalentsto 13.0 equivalents relative to the fatty acids in the raw materialcomposition.

[14] The manufacturing method according to any one of [8] to [13], wherean amount of the alkali catalyst in the reaction solution is from 1.0equivalent to 2.3 equivalents relative to the fatty acids in the rawmaterial composition.

[15] The manufacturing method according to any one of [8] to [14], wherethe alkali catalyst is at least one selected from the group consistingof sodium hydroxide and potassium hydroxide.

[16] The manufacturing method according to any one of [8] to [15], wherethe temperature condition of the hydrolysis treatment is from −20° C. to10° C.

[17] The manufacturing method according to any one of [8] to [16], wherethe raw material composition is derived from a microbial raw material.

[18] A food product, supplement, medicament, cosmetic, or animal feedcontaining the free polyunsaturated fatty acid-containing compositiondescribed in any one of [1] to [7].

[19] Use of the free polyunsaturated fatty acid-containing compositiondescribed in any one of [1] to [7] in a manufacturing method of a foodproduct, supplement, medicament, cosmetic, or animal feed.

According to aspects of the present disclosure, a free polyunsaturatedfatty acid composition having less impurities and a manufacturing methodfor a free polyunsaturated fatty acid composition having less impuritiescan be provided.

DETAILED DESCRIPTION

The free polyunsaturated fatty acid-containing composition according toan aspect of the present disclosure is a free polyunsaturated fattyacid-containing composition containing at least one free polyunsaturatedfatty acid having 20 or more carbons in an amount that a content thereofis 80.0% or greater of fatty acids in the composition, and the freepolyunsaturated fatty acid-containing composition satisfies at least oneselected from the group consisting of conditions (1) and (2):

(1) a content of a conjugated unsaturated fatty acid being 1.0% or lessof the fatty acids in the composition; and

(2) the Gardner color being less than 3+.

The manufacturing method of a free polyunsaturated fatty acid accordingto an aspect of the present disclosure is a manufacturing method of afree polyunsaturated fatty acid-containing composition, the methodincluding:

providing a raw material composition containing at least onepolyunsaturated fatty acid having 20 or more carbons; and

performing hydrolysis treatment on a reaction solution containing theprovided raw material composition, a lower alcohol, water, and an alkalicatalyst in a temperature condition at 10° C. or lower.

A composition containing a free long-chain polyunsaturated fatty acidhaving 20 or more carbons has higher polarity compared to the polarityof a long-chain polyunsaturated fatty acid in an alkyl ester form orglyceride form and may exhibit different behavior from the behavior ofthe alkyl ester form or the glyceride form. In particular, conjugatedunsaturated fatty acid or a coloring substance may be increased as animpurity during a hydrolysis step by an alkali catalyst to obtain a freepolyunsaturated fatty acid in high concentration. It was found that,unlike the case of a composition containing a long-chain polyunsaturatedfatty acid in an alkyl ester form or glyceride form, these impuritiesare difficult to separate or to be removed from the compositioncontaining a free long-chain polyunsaturated fatty acid in highconcentration. Meanwhile, it was also found that these impurities aregenerated or increased due to heat in the hydrolysis step, andgeneration or increase in these substances can be suppressed by settingthe temperature condition of the hydrolysis step to a particular range.

That is, in the free polyunsaturated fatty acid-containing composition,at least one of the content of the conjugated unsaturated fatty acid orthe content of the coloring substance is lower than the content of thecase where the hydrolysis treatment with an alkali catalyst to obtain afree fatty acid is performed at a conventional temperature condition. Itwas unexpected that each of the content of the conjugated unsaturatedfatty acid and the content of the coloring substance can be adjusted bysetting the temperature condition in the hydrolysis treatment to obtaina fatty acid in a free form to a specific range, and can be reducedcompared to each content before the hydrolysis treatment. For example,the content of at least one selected from the group consisting of theconjugated unsaturated fatty acid and the coloring substance, which isincreased when the temperature condition of typical hydrolysis treatmentusing an alkali catalyst, such as 70° C., is applied, can be reduced bylowering the temperature condition of the hydrolysis treatment to aspecific range.

Accordingly, the content of the conjugated unsaturated fatty acid and/orthe coloring substance that is generated or increased in the hydrolysisstep is small as well as the content of at least one of freepolyunsaturated fatty acid having 20 or more carbons, which is thetarget, is high, in this free polyunsaturated fatty acid-containingcomposition. Therefore, the content of these particular impurities issmall, and better function of the at least one of free polyunsaturatedfatty acid having 20 or more carbons, which is the target, can befavorably exhibited.

The manufacturing method of this free polyunsaturated fattyacid-containing composition includes performing the hydrolysis treatmenton the raw material composition in a temperature condition of 10° C. orlower. As a result, the content of the particular impurities describedabove is small, and the composition containing at least one freepolyunsaturated fatty acid having 20 or more carbons, which is thetarget, can be efficiently obtained.

The terms “oil” and “oil and fat” in the present specification includeoils containing only triglycerides, and also include crude oilscontaining triglycerides as a main component and other lipids such asdiglycerides, monoglycerides, phospholipids, cholesterol, and free fattyacids. “Oil” and “oil and fat” mean compositions containing theselipids.

The term “fatty acid” not only indicates a free saturated or unsaturatedfatty acid itself, but also includes fatty acids contained asconstituent units in free saturated or unsaturated fatty acids,saturated or unsaturated fatty acid alkyl esters, triglycerides,diglycerides, monoglycerides, phospholipids, steryl esters, and thelike, which can also be called constituent fatty acids. In the presentspecification, unless otherwise noted or indicated, when a fatty acidthat is present or used is mentioned, presence or use of fattyacid-containing compounds in any form is included. Examples of forms ofcompounds containing fatty acids include a free fatty acid form, a fattyacid alkyl ester form, a glyceryl ester form, a phospholipid form, and asteryl ester form. When a fatty acid is specified, one form may bepresent, or a mixture of two or more forms may be present.

It has been empirically determined that the reaction efficiency ofhydrolysis of fatty acids is high, and after hydrolysis, a compositioncontaining mainly fatty acid in a form of free fatty acids is obtained.For this reason, unless otherwise specified, fatty acids after theprocessing step may be denoted while omitting that they are acomposition or that the fatty acid is of a free fatty acid form.However, this does not completely negate the fact that fatty acids in aform other than a free fatty acid form are included.

It has been empirically determined that the reaction efficiency ofalcoholysis of oils and fats or fatty acid esters is high, and afteralcoholysis, a composition containing mainly fatty acid in a fatty acidalkyl ester form is obtained. For this reason, unless otherwisespecified, fatty acids after the processing step are denoted whileomitting that they are a composition or that the fatty acid is in analkyl ester form. However, this does not completely negate the fact thatfatty acids in a form other than an alkyl ester form are included.

When denoting fatty acids, a numerical expression may be used, wherebythe number of carbons, the number of double bonds, and the locations ofdouble bonds are each expressed in a simplified manner using numbers andalphabets, respectively. For example, a saturated fatty acid having 20carbons is denoted as “C20:0”. A monounsaturated fatty acid having 18carbons is denoted as “C18:1” or the like. Dihomo-γ-linolenic acid isdenoted as “C20:3, n-6” or the like. Note that “n-6” is denoted also asω-6, and this indicates that the bonding position of a first double bondis at the sixth position when the position is counted from the lastcarbon (ω) to the carboxy group. This method is known to those ofordinary skill in the art, and those of ordinary skill in the art caneasily specify fatty acids expressed in accordance with this method.

In the present specification, the term “crude oil” means a mixture ofthe lipids described above, and means an oil in the state obtained byextraction from an organism. In the present specification, the term“refined oil” means an oil from which substances, such as phospholipidsand cholesterol, other than the target substance which have been removedby performing at least one oil and fat refining process selected fromthe group consisting of a degumming process, a deacidification process,a decoloring process, and a deodorizing process.

In the present specification, in addition to an independent step, theterm “step” also refers to a step that achieves an intended object ofthe step even when the step cannot be clearly distinguished from othersteps.

In the present specification, numeric ranges indicated by “to” areranges that include the minimum and maximum values each stated beforeand after the “to.” In the present specification, the terms “not greaterthan” and “less than” in regard to percentages mean ranges including 0%,which is the case of “not contained”, or a value undetectable by presentmeans, unless the lower limit is specifically stated.

In the present specification, in a case where multiple substancescorresponding to each of the components in the composition are present,the amount of each component in the composition, unless otherwise noted,is taken to mean the total amount of these multiple substances presentin the composition. In the present specification, in the case wheremultiple substances corresponding to each of the components in thecomposition are present, the content of each component in thecomposition, unless otherwise noted, is taken to mean the total contentof these multiple substances present in the composition.

In the present specification, unless otherwise noted, when a numericalrange that only specifies one or a plurality of upper limit values and anumerical range that only specifies one or a plurality of lower limitvalues are described for an identical target, an embodiment of thepresent disclosure includes a numerical range having a combination ofany upper limit value that is chosen from the one or the plurality ofthe upper limit values and any lower limit value that is chosen from theone or the plurality of the lower limit values.

The content of the fatty acids in the composition of the presentspecification is determined based on the fatty acid composition unlessotherwise noted. The composition of fatty acids may be determined by anormal method. Specifically, when the fatty acids in the composition tobe measured are substances other than fatty acid lower alkyl esters,fatty acid lower alkyl esters, which is obtained by subjecting the fattyacids to be measured to esterification by using a lower alcohol and acatalyst, are used. When the fatty acids in the composition to bemeasured are fatty acid lower alkyl esters, the fatty acids to bemeasured are used as is. Thereafter, the obtained fatty acid lower alkylesters are analyzed as a sample using gas chromatography. Peakscorresponding to each of the fatty acids are identified in the obtainedgas chromatography chart, and the peak area of each of the fatty acidsis determined using the Agilent ChemStation integration algorithm(revision C.01.03[37], Agilent Technologies). “Peak area” indicates aratio (area percent) of the peak area for respective components to thearea of all peaks as determined in charts analyzed by gaschromatography, thin-layer chromatography/flame ionization detector(TLC/FID) or the like of oil and fat having various fatty acids asconstituent components, and indicates the content ratio of the componentof the peak. The value according to the area percent obtained by themeasurement method described above is the same as the value according tothe weight percent of each fatty acid relative to the total weight ofthe fatty acids in a sample, and may be used interchangeably. Refer to“Basic Oil Analytical Test Methods”, 2013 Edition, 2.4.2.1-2013 FattyAcid Composition (FID constant temperature gas chromatograph method) and2.4.2.2-2013 Fatty Acid Composition (FID heating gas chromatographmethod) established by the Japan Oil Chemists' Society (JOCS).

The fatty acid composition was determined by gas chromatography by themethod indicated in the examples. Detailed conditions are indicated inexamples.

Free Polyunsaturated Fatty Acid-Containing Composition

The free polyunsaturated fatty acid-containing composition according toan embodiment of the present disclosure is a free polyunsaturated fattyacid-containing composition containing at least one free polyunsaturatedfatty acid having 20 or more carbons in an amount that a content thereofis 80.0% or greater of fatty acids in the composition, and the freepolyunsaturated fatty acid-containing composition satisfies at least oneselected from the group consisting of conditions (1) and (2) below:

(1) the content of a conjugated unsaturated fatty acid being 1.0% orless of the fatty acids in the composition: and

(2) the Gardner color being less than 3+.

The free polyunsaturated fatty acid-containing composition satisfies atleast one selected from the group consisting of the conditions (1) and(2) and contains at least 80.0% of at least one free polyunsaturatedfatty acid having 20 or more carbons. As a result, the amount ofparticular impurities is small, and better functions of the at least onefree polyunsaturated fatty acid having 20 or more carbons can befavorably exhibited.

In the present specification, unless otherwise noted, the freepolyunsaturated fatty acid having 20 or more carbons may be referred toas “free LC-PUFA”. In the present specification, the freepolyunsaturated fatty acid-containing composition according to anembodiment of the present disclosure may be simply referred to as “freeLC-PUFA-containing composition”.

The polyunsaturated fatty acid having 20 or more carbons in the freeLC-PUFA-containing composition includes di- or higher valent unsaturatedfatty acids and, preferably, tri- or higher valent unsaturated fattyacids. The number of carbon atoms of the polyunsaturated fatty acidrefers to the number of carbon atoms of the constituent fatty acids.Examples of polyunsaturated fatty acid having 20 or more carbons includepolyunsaturated fatty acids having from 20 to 22 carbons. Specificexamples thereof include eicosadienoic acid (C20:2, n-9, EDA),dihomo-γ-linolenic acid (C20:3, n-6, DGLA), Mead acid (C20:3, n-9, MA),eicosatetraenoic acid (C20:4, n-3, ETA), arachidonic acid (C20:4, n-6,ARA), eicosapentaenoic acid (C20:5, n-3, EPA), docosatetraenoic acid(C22:4, n-6, DTA), docosapentaenoic acid (C22:5, n-3, _(n-3)DPA),docosapentaenoic acid (C22:5, n-6, _(n-6)DPA), and docosahexaenoic acid(C22:6, n-3, DHA). The free LC-PUFA-containing composition needs tocontain at least one of these polyunsaturated fatty acids and maycontain a combination of two or more of these. Examples of the LC-PUFAhaving a combination of two or more of these include a combination ofDGLA and EPA, a combination of DGLA and _(n-3)DPA, a combination of DGLAand DHA, a combination of ARA and EPA, a combination of ARA and_(n-3)DPA, a combination of ARA and DHA, a combination of EPA and_(n-3)DPA, a combination of DHA and _(n-3)DPA, a combination of DHA andEPA, and a combination of EPA and DHA and _(n-3)DPA.

The free LC-PUFA-containing composition contains one selected from thepolyunsaturated fatty acids described above and may contain no otherpolyunsaturated fatty acids, or does not need to contain otherparticular one or two or more polyunsaturated fatty acids describedabove as long as the free LC-PUFA-containing composition contains atleast one polyunsaturated fatty acid having from 20 to 22 carbonsdescribed above as LC-PUFA. For example, the free LC-PUFA-containingcomposition may be prepared so as to not contain at least one typeselected from the group consisting of eicosadienoic acid (C20:2, n-9),dihomo-γ-linolenic acid (C20:3, n-6), Mead acid (C20:3, n-9),eicosatetraenoic acid (C20:4, n-3), arachidonic acid (C20:4, n-6),eicosapentaenoic acid (C20:5, n-3), docosatetraenoic acid (C22:4, n-6),docosapentaenoic acid (C22:5, n-3), docosapentaenoic acid (C22:5, n-6),and docosahexaenoic acid (C22:6, n-3). Here, “not containingpolyunsaturated fatty acids” means that the content of the targetpolyunsaturated fatty acid is less than 5% or 0% of the fatty acids inthe composition.

The content of the LC-PUFA in the free LC-PUFA-containing composition is80.0% or greater of the fatty acids in the composition. Because the freeLC-PUFA-containing composition containing 80.0% or greater of theLC-PUFA can exhibit superior functions of LC-PUFA. The lower limit valueof the content of the target LC-PUFA in the free LC-PUFA-containingcomposition may be 85.0%, 90.0%, 95.0%, 97.0%, 98.0%, 99.0%, or 99.5% ofthe fatty acids in the composition. When the content of the LC-PUFA ishigher, superior functions of the LC-PUFA can be exhibited. The upperlimit value of the content of the LC-PUFA is not particularly limitedand, for example, the upper limit value may be 99.9% or 98.0%. Thecontent of the LC-PUFA in the present composition may be in a range ofany combination of a chosen value of the upper limit value and a chosenvalue of the lower limit value described above. For example, the contentmay be from 80.0% to 99.9%, from 90.0% to 99.9%, from 90.0% to 98%, from95.0% to 99.9%, from 97.0% to 99.9%, or from 97.0% to 98.0% of the fattyacids in the composition.

The free LC-PUFA-containing composition satisfies at least one selectedfrom the group consisting of the conditions (1) and (2).

The condition (1) that may be satisfied by the free LC-PUFA-containingcomposition relates to the content of the conjugated unsaturated fattyacid. The content of the conjugated unsaturated fatty acid is 1.0% orless of the fatty acids in the composition. Although the conjugatedunsaturated fatty acid differs depending on the type of the fatty acidand the type of the LC-PUFA in the raw material composition used for thehydrolysis treatment, examples of the conjugated unsaturated fatty acidinclude conjugated dienoic acid, conjugated trienoic acid, andconjugated tetraenoic acid. The conjugated unsaturated fatty acid can bequantified based on the absorbance of the target conjugated unsaturatedfatty acid. The content of the conjugated unsaturated fatty acid in thefree LC-PUFA-containing composition is a content of the conjugatedunsaturated fatty acid obtained by measuring ultraviolet spectrum of asample and calculating using a stipulated calculation formula, and is avalue measured in accordance with the conjugated unsaturated fatty acid(spectrum method) stipulated in Reference 1.14 of Standard Methods forthe Analysis of Fats, Oils, and Related Materials, 2013 Edition,established by Japan Oil Chemists' Society (JOCS). When the compositionin the sample contains a component other than the fatty acids, theamount of the conjugated unsaturated fatty acid can be determined basedon the amount of the fatty acids in the composition.

The content of the conjugated unsaturated fatty acid in the freeLC-PUFA-containing composition may be 0.8% or less, 0.7% or less, 0.6%or less, 0.5% or less, 0.4% or less, or 0.3% or less, of the fatty acidsin the composition. A smaller content of the conjugated unsaturatedfatty acid tends to exhibit superior oxidation stability of thecomposition. The lower limit value of the content of the conjugatedunsaturated fatty acid may be 0.1%, 0.2%, 0.01%, or 0.001%. For example,the content of the conjugated unsaturated fatty acid of the freeLC-PUFA-containing composition may be from 0.001% to 1.0%, from 0.01% to0.8%, from 0.1% to 0.7%, or from 0.2% to 0.7%, of the fatty acids in thecomposition.

The condition (2) that may be satisfied by the free LC-PUFA-containingcomposition relates to the Gardner color and is the Gardner color ofless than 3+. The Gardner color is an indicator varied based on thecontent of the coloring substance present in the free LC-PUFA-containingcomposition and indicates that the degree of coloration of thecomposition is increased in the following order: 1−, 1, 1+, 2−, 2.2+.3−, 3.3+. 4−, 4, 4+, 5−, 5, 5+. Satisfying the condition (2) means thatthe color of the free LC-PUFA-containing composition is the Gardnercolor of less than 3+, that is, the color corresponds to any one of 1−,1, 1+, 2−, 2, 2+, 3−, or 3. The Gardner color is determined based on3.2.1.1 of the Standard Methods for the Analysis of Fats, Oils, andRelated Materials, 2013 Edition, established by Japan Oil Chemists'Society (JOCS).

The Gardner color of the free LC-PUFA-containing composition may be 3−or less, 2+ or less, 2 or less, 1+ or less, or 1−. The freeLC-PUFA-containing composition exhibiting a lower value of the Gardnercolor has a smaller amount of the coloring substance that may begenerated due to hydrolysis using an alkali catalyst and tends tofacilitate quality control and tends to provide a wider selection forcommercialization.

The free LC-PUFA-containing composition can satisfy one of the condition(1) or (2). That is, when the free LC-PUFA-containing compositionsatisfies the condition (1), the Gardner color may be 3+ or greater, andin this case, for example, the Gardner color may be 4 or less or 4− orless. When the free LC-PUFA-containing composition satisfies thecondition (2), the content of the conjugated unsaturated fatty acid maybe greater than 1.0%, and in this case, for example, the content may be3.0% or less, 2.5% or less, 2.0% or less, or 1.5% or less.

In the free LC-PUFA-containing composition, the condition (3) may be ananisidine value (AnV) of 5.0 or less, 4.5 or less, 4.0 or less, 3.5 orless, 3.0 or less, or 2.5 or less. The anisidine value is an indicatorvaried based on the content of the oxidized substance present in thefree LC-PUFA-containing composition. The free LC-PUFA-containingcomposition showing a lower anisidine value has a smaller content of theoxidized substance. The anisidine value is determined based on 2.5.3 ofthe Standard Methods for the Analysis of Fats, Oils, and RelatedMaterials, 2013 Edition, established by Japan Oil Chemists' Society(JOCS).

There can be cases where the free LC-PUFA-containing composition onlysatisfies the condition (1), where the free LC-PUFA-containingcomposition only satisfies the condition (2), or where the freeLC-PUFA-containing composition satisfies the conditions (1) and (2).Furthermore, in addition to these, the condition (3) may be satisfied.The free LC-PUFA-containing composition includes the following, forexample:

(a) a free LC-PUFA-containing composition having the content of theconjugated unsaturated fatty acid of 1.0% or less of the fatty acids inthe composition and having the Gardner color of 3+ or greater;

(b) a free LC-PUFA-containing composition having the content of theconjugated unsaturated fatty acid of greater than 1.0% of the fattyacids in the composition and having the Gardner color of 3 or less;

(c) a free LC-PUFA-containing composition having the content of theconjugated unsaturated fatty acid of 1.0% or less of the fatty acids inthe composition and having the Gardner color of 3 or less;

(d) a free LC-PUFA-containing composition having the content of theconjugated unsaturated fatty acid of 1.0% or less of the fatty acids inthe composition, having the Gardner color of 3+ or greater, and havingthe anisidine value of 5.0 or less;

(e) a free LC-PUFA-containing composition having the content of theconjugated unsaturated fatty acid of greater than 1.0% of the fattyacids in the composition, having the Gardner color of 3 or less, andhaving the anisidine value of 5.0 or less: and

(f) a free LC-PUFA-containing composition having the content of theconjugated unsaturated fatty acid of 1.0% or less of the fatty acids inthe composition, having the Gardner color of less than 3+, and havingthe anisidine value of 5.0 or less.

In the free LC-PUFA-containing compositions of (a) to (c) describedabove, the anisidine value may be greater than 5.0 and may be 6.0 orless, or 5.5 or less. In the free LC-PUFA-containing compositions of (a)and (d) described above, the Gardner color may be 3+ or greater and maybe 6 or less, 5+ or less, 5 or less, 5− or less, 4+ or less, 4 or less,or 4− or less. In the free LC-PUFA-containing compositions of (b) and(e) described above, the content of the conjugated unsaturated fattyacid may be 2.5% or less, 2.0% or less, or 1.5% or less.

The free LC-PUFA-containing composition of (f) described above includesthe following, for example:

(f1) a free LC-PUFA-containing composition having the content of theconjugated unsaturated fatty acid of 0.7% or less, having the Gardnercolor of 2+ or less, and having the anisidine value of 5.0 or less;

(f2) a free LC-PUFA-containing composition having the content of theconjugated unsaturated fatty acid of 0.7% or less, having the Gardnercolor of less than 3+, and having the anisidine value of 4.0 or less;

(f3) a free LC-PUFA-containing composition having the content of theconjugated unsaturated fatty acid of 0.7% or less, having the Gardnercolor of 2+ or less, and having the anisidine value of 4.0 or less;

(f4) a free LC-PUFA-containing composition having the content of theconjugated unsaturated fatty acid of 0.4% or less, having the Gardnercolor of 1+ or less, and having the anisidine value of 3.5 or less;

(f5) a free LC-PUFA-containing composition having the content of theconjugated unsaturated fatty acid of 0.4% or less, having the Gardnercolor of 1 or less, and having the anisidine value of 2.5 or less; and

(f6) a free LC-PUFA-containing composition having the content of theconjugated unsaturated fatty acid of 0.3% or less, having the Gardnercolor of 1−, and having the anisidine value of 3.5 or less.

In the free LC-PUFA-containing compositions of (a) to (f) describedabove, the content of the conjugated unsaturated fatty acid may be0.001% or greater, 0.01% or greater, or 0.1% or greater.

The free LC-PUFA-containing composition may have a small content of thefatty acid alkyl ester. In the step of producing a free fatty acid, thefatty acid alkyl ester may be a raw material substance of alkalinehydrolysis or may be a product that can be produced from the free fattyacid through a reverse reaction. The free LC-PUFA-containing compositionhaving a smaller content of the fatty acid alkyl ester can have a highercontent of the free LC-PUFA and tends to exhibit superiorbioabsorbability, especially superior intestinal absorbability, of thecomposition. The content of the fatty acid alkyl ester of the freeLC-PUFA-containing composition may be 0.2% or less, 0.1% or less, 0.05%or less, 0.04% or less, 0.03% or less, 0.02% or less, or 0.01% or less,of the fatty acids in the composition. The lower limit value of thecontent of the fatty acid alkyl ester is not particularly limited, andfor example, the lower limit value may be 0.0005%. When the content ofthe fatty acid alkyl ester is 0.0005% or greater, the composition isless likely to crystallize, and the flowability tends to be enhanced.

The free LC-PUFA-containing composition may be a free LC-PUFA-containingcomposition having a smaller content of fatty acids other than theLC-PUFA. When the content of the fatty acids other than the LC-PUFA inthe composition is low, exhibition of functions is expected in a degreecorresponding to the content of the LC-PUFA, and also effect due toother fatty acids other than the LC-PUFA can be suppressed. Examples ofother fatty acids that can reduce the content thereof in the freeLC-PUFA-containing composition include saturated or unsaturated fattyacids having less than 20 carbons, and saturated fatty acids having 22or more carbons. Specific examples of the saturated or unsaturated fattyacid having less than 20 carbons include saturated fatty acids having 18carbons, monounsaturated fatty acids having 18 carbons, divalentunsaturated fatty acids having 18 carbons, trivalent unsaturated fattyacids having 18 carbons, and tetravalent unsaturated fatty acids having18 carbons. Examples of the saturated fatty acid having 22 or morecarbons include saturated fatty acids having 22 carbons and saturatedfatty acids having 24 carbons.

Among these fatty acids other than the LC-PUFA, the freeLC-PUFA-containing composition may be a free LC-PUFA-containingcomposition having a low content of a di- or higher-valentpolyunsaturated fatty acid having 18 carbons. For example, the contentof the di- or higher-valent polyunsaturated fatty acid having 18 carbonsmay be 2.0% or less, 1.5% or less, 1.0% or less, or 0.8% or less, of thefatty acids in the composition. The lower limit value of the content ofthe fatty acids other than the LC-PUFA may be, for example, 0.001% orgreater, 0.005% or greater, or 0.01%. The content of the di- orhigher-valent polyunsaturated fatty acid having 18 carbons may be, forexample, from 0.001% to 2.0%, from 0.005% to 1.5%, from 0.01% to 1.5%,or from 0.01% to 1.0%.

The free LC-PUFA-containing composition may contain a fatty acid in aform other than the fatty acids described above. Examples of the fattyacids in other forms include triglyceride, diglyceride, monoglyceride,phospholipid, and steryl esters. The content of the fatty acids in otherforms needs to be an amount that corresponds to the rest of the freeLC-PUFA-containing composition excluding the LC-PUFA. The content may beless than 20.0%, less than 10.0%, less than 5.0%, less than 2.0%, lessthan 1.0%, or less than 0.5%, of the fatty acids in the composition.

The content of the fatty acids in the free LC-PUFA-containingcomposition may be 97.0 wt. % or greater, 98.0 wt. % or greater, 99.0wt. % or greater, 99.5 wt. % or greater, or 100 wt. %, of the totalweight of the composition. The content of the fatty acids in the freeLC-PUFA-containing composition can be confirmed by a publicly knowntechnique, such as TLC/FID. The free LC-PUFA-containing composition maycontain a component other than the fatty acids. Examples of such othercomponent that may be contained in the free LC-PUFA-containingcomposition include antioxidants, such as tocopherol, vitamin C, andvitamin C derivatives, and solvents, such as ethanol.

The free LC-PUFA-containing composition may be produced by anymanufacturing method as long as the free LC-PUFA-containing compositionhas characteristics described in the present specification, andpreferably is a free LC-PUFA-containing composition produced by themanufacturing method described below.

Manufacturing Method

The manufacturing method of the free LC-PUFA-containing composition inan embodiment of the present disclosure includes: providing a rawmaterial composition containing at least one polyunsaturated fatty acidhaving 20 or more carbons; and performing hydrolysis treatment on areaction solution containing the provided raw material composition, alower alcohol, water, and an alkali catalyst in a temperature conditionat 10° C. or lower: and, as necessary, other step(s). According to thepresent manufacturing method, a free LC-PUFA-containing compositionhaving a lower content of the conjugated unsaturated fatty acid and/orthe coloring substance than the content of the case where hydrolysistreatment is performed at a temperature of higher than 10° C. can beefficiently obtained.

In the step of providing the raw material composition, a raw materialcomposition that had been acquired may be provided or a raw materialcomposition that had been separately produced may be provided as long asthe raw material composition contains at least one LC-PUFA. The rawmaterial composition may be a raw material composition derived from anorganism, such as a raw material composition derived from a marine rawmaterial, a raw material composition derived from a microbial rawmaterial, a raw material composition derived from a plant raw material,and a raw material composition derived from an animal raw material. Theraw material composition may be a composition containing LC-PUFA in atriglyceride form, and may be a composition containing an LC-PUFA alkylester. The LC-PUFA alkyl ester-containing composition is preferablyobtained by subjecting a bio-oil containing LC-PUFA in a triglycerideform to alkyl esterification.

The bio-oil containing LC-PUFA may be a bio-oil, such as a marine rawmaterial oil derived from fish or the like, a microbial oil derived frommicroorganisms, and a plant oil derived from plants, and for example,may be a microbial oil. The bio-oil means an oil obtained by usingbiomass as its origin, and the microbial oil means an oil obtained byusing microbial biomass as its origin. The bio-oil may be a bio-oil thatoriginates from genetically modified materials. The term “biomass” meansan aggregation or lump of cells at a certain point of time during growthin a certain region or in an ecosystem.

Examples of the marine raw material oil include lipids including oilsand fats, phospholipids, wax esters, and the like contained in fish,shellfish, or marine animals. Examples of the marine raw material oilinclude oils derived from fish such as herring, sardine, anchovy,menhaden, pilchard, saury, tuna, bonito, hake, catfish, capelin, redfish, white fish, mackerel, jack mackerel, yellowtail, sand eel, pout,salmon, pollock, cod, halibut, trout, blue whitening, sprat, shark, anddogfish; oils derived from mollusks such as squid, clam, and abalone:oils derived from crustaceans such as krill; oils derived from animalssuch as seal, sealion, sea bear, and walrus, and mixtures of these oils.

The microorganism may be a microorganism that produces lipids or amicroorganism that can produce lipids, and examples thereof includealgae, true fungi, bacteria, fungi, and stramenopiles.

Examples of the algae include the genus Labyrinthula (Labyrinthulamycota).

Examples of the true fungi include the genus Yarrowia, the genusCandida, the genus Saccharomyces, the genus Schizosaccharomyces, and thegenus Pichia.

Examples of the bacteria include Agrobacterium, Bacillus, Escherichia,Pseudomonas, and Actinomyces.

Examples of the fungi include at least one type selected from the groupconsisting of the genus Mortierella, the genus Conidiobolus, the genusPhythium, the genus Phytophthora, the genus Penicillium, the genusCladosporium, the genus Mucor, the genus Fusarium, the genusAspergillus, the genus Rhodotorula, the genus Entomophthora, the genusEchinosporangium, and the genus Saprolegnia. Of these, microorganismsbelonging to the genus Mortierella are even more preferable. Examples ofthe microorganisms belonging to the genus Mortierella includemicroorganisms belonging to the subgenus Mortierella such as Mortierellaelongata, Mortierella exigua, Mortierella hygrophila, and Mortierellaalpina.

Examples of the plant include plants of the genus Brassica, the genusHelianthus, the genus Gossypium, the genus Linum, the genus Nicotiana,the genus Citrus, the genus Allium, the genus Triticum, the genusHordeum, the genus Avena, the genus Secale, the genus Oryza, the genusSaccharum, the genus Zea, the genus Sorghum as well as soybean, tomato,potato, pea, frijol, peanut, Medicago, celery, paseley, clover, carrot,radish, sugar beet, cucumber, spinach, cassava, olive, apple, banana,melon, grape, strawberry, coconut plant, coffee plant, and pepper.

The raw material oil that is subjected to the alkyl esterification maybe a crude oil or a refined oil. The crude oil may be an oil obtainedfrom a marine raw material or may be an oil obtained from a microbialraw material. A refined oil can be obtained by subjecting a crude oil toa de-gumming process, deacidification process, decoloration processusing an activated clay or active carbon, washing process, deodorizationprocess by steam distillation or the like, and crude oil refiningprocess that removes substances other than the target, such asphospholipids and sterols.

In the step of performing alkyl esterification, the raw material oil isdecomposed into a lower alkyl ester via alcoholysis using a loweralcohol. Examples of the lower alcohol include lower alcohols typicallyused in alkyl esterification of fatty acids, such as lower alcoholshaving from 1 to 3 carbons. In the alcoholysis, a lower alcohol such asethanol and a catalyst or enzyme are added and reacted with a rawmaterial oil to produce an ethyl ester from the fatty acid bonded toglycerin. As the catalyst, an alkali catalyst, an acid catalyst, or thelike is used. As the enzyme, lipase is used.

The crude oil or the refined oil, or the fatty acid alkylester-containing composition obtained by the alkyl esterificationtreatment may contain at least one other fatty acid in addition to thetarget LC-PUFA. One type of method or a combination of two or more typesof methods, exemplified by the distillation, rectification, columnchromatography, low temperature crystallization method, urea clathratemethod, liquid-liquid countercurrent distribution chromatography, or thelike, may be used to concentrate or isolate the particular LC-PUFA fromthe crude oil, the refined oil, or the fatty acid alkyl ester-containingcomposition. A combination of distillation or rectification, and columnchromatography or liquid-liquid countercurrent distributionchromatography is preferably used. When the step of concentrating orisolating the particular LC-PUFA is performed, the content of targetLC-PUFA, which may be contained in the final LC-PUFA-containingcomposition, in the fatty acids is increased and the content of otherfatty acid other than the target LC-PUFA in the fatty acids can bereduced.

For example, in a case in which rectification is used, the rectificationstep is preferably carried out by distillation using a reduced pressureat the top of the distillation column of less than or equal to 10 mmHg(1333 Pa), using a temperature of the column bottom in the range of 165°C. to 210° C., and preferably 170° C. to 195° C., from the perspectiveof suppressing the denaturation of the fatty acid due to heat, andincreasing efficiency of rectification. The pressure at the top of thedistillation column is preferably as low as possible, and morepreferably lower than or equal to 0.1 mmHg (13.33 Pa). No particularlimitation is imposed on the temperature at the top of the column, andfor example, this temperature may be set to lower than or equal to 160°C. In the rectification step, a raw material composition having an evenhigher content of the LC-PUFA, such as LC-PUFA alkyl ester, may beobtained.

Reverse phase distribution type column chromatography is preferred asthe column chromatography. The reverse phase column chromatography maybe reverse phase column chromatography that is known in the art, andhigh-performance liquid chromatography (HPLC) using a base materialmodified with octadecylsilyl groups (ODS) as a stationary phase isparticularly preferable.

The composition obtained by the concentration or isolation step is acomposition having a high content of the target LC-PUFA and, forexample, the content of the target LC-PUFA may be 80.0% or greater,85.0% or greater, 90.0% or greater, 95.0% or greater, 97.0% or greater,98.0% or greater, 99.0% or greater, or 99.5% or greater, of the fattyacids. This composition containing a high concentration of LC-PUFA canbe used as a raw material composition.

In the step of performing hydrolysis treatment, a reaction solutioncontaining the provided raw material composition, a lower alcohol,water, and an alkali catalyst is subjected to hydrolysis treatment in atemperature condition at 10° C. or lower. In the present specification,this hydrolysis treatment with an alkali catalyst may be referred to asalkali hydrolysis treatment.

The reaction solution used in the alkali hydrolysis treatment contains araw material composition, a lower alcohol, water, and an alkalicatalyst. The raw material composition may be a bio-oil or may be anLC-PUFA alkyl ester-containing composition. The concentration (w/w) ofthe raw material composition in the reaction solution may be from 10.0wt. % to 70.0 wt. %, from 20.0 wt. % to 60.0 wt. %, or from 40 wt. % to50 wt. %, from the perspective of reaction efficiency.

Examples of the lower alcohol include lower alcohols typically used fordecomposing bio-oils or fatty acid alkyl esters to obtain free fattyacids, such as lower alcohols having from 1 to 3 carbons. The amount ofthe lower alcohol in the reaction solution needs to be an amount that iseffective in decomposing a fatty acid in the raw material compositioninto a free fatty acid. For example, the amount may be from 0.9equivalents to 32.0 equivalents, from 0.92 equivalents to 20.0equivalents, from 0.95 equivalents to 14 equivalents, from 2.0equivalents to 10.0 equivalents, from 3.0 equivalents to 7.0equivalents, or from 4.5 equivalents to 5.5 equivalents, relative to theamount of the fatty acids in the composition. When the ratio of thelower alcohol to the fatty acids in the raw material composition is 0.9equivalents or greater, the reaction tends to proceed at a morefavorable rate, and suppression of generation of the coloring substancetends to be facilitated. On the other hand, when the ratio is 32.0equivalents or less, the condition after the termination of the reactiontends to be stabilized, and progression of reverse reaction that maygenerate fatty acid alkyl esters tends to be effectively suppressed. Theamount of the lower alcohol in the reaction solution includes both theamount of the lower alcohol added during the preparation of the reactionsolution and the amount of the lower alcohol that is produced during thereaction as a byproduct in the reaction solution. In the presentspecification, “equivalent” refers to “molar equivalent”. This is thesame hereafter.

The amount of the lower alcohol in the reaction solution may be from0.20 to 8.20, from 0.23 to 4.50, from 0.25 to 3.50, from 0.60 to 2.50,or from 1.20 to 1.50, in terms of weight ratio relative to the amount ofwater. When the weight ratio of lower alcohol to water is in this range,the alkali hydrolysis proceeds even more favorably, the condition afterthe termination of the reaction tends to be stabilized, and progressionof reverse reaction that may generate fatty acid alkyl esters tends tobe effectively suppressed. The amount of the lower alcohol in thereaction solution includes both the amount of the lower alcohol addedduring the preparation of the reaction solution and the amount of thelower alcohol that is produced during the reaction as a byproduct in thereaction solution.

The amount of the reaction solution in water may be from 6.0 equivalentsto 13.0 equivalents, from 7.0 equivalents to 12.0 equivalents, from 8.0equivalents to 11.0 equivalents, or from 9.0 equivalents to 10.0equivalents, relative to the amount of the fatty acids in the rawmaterial composition. When the weight ratio of water to raw materialcomposition is in this range, the alkali hydrolysis can be morefavorably proceeded.

The alkali catalyst used in the alkali hydrolysis treatment may be analkali metal hydroxide, may be sodium hydroxide, potassium hydroxide, orthe like, may be at least one selected from the group consisting ofsodium hydroxide and potassium hydroxide, and is more preferably sodiumhydroxide. The amount of the alkali catalyst used in the alkalihydrolysis treatment needs to be in a range that can produce a freefatty acid from the raw material composition. For example, the amountmay be from 1.0 equivalent to 2.3 equivalents, from 1.0 equivalent to2.0 equivalents, or from 1.0 equivalent to 1.5 equivalents, relative tothe amount of the fatty acids in the raw material composition. When theratio of the alkali catalyst to the raw material composition is in thisrange, reaction can be efficiently proceeded to obtain the free LC-PUFA.

The reaction solution may contain a component other than the substancesdescribed above in the range that does not impair progression of thealkali hydrolysis reaction. Examples of the component includeantioxidants, such as tocopherol, vitamin C, and vitamin C derivatives,and non-alcohol solvents, such as acetone.

The hydrolysis treatment in this manufacturing method is performed in atemperature condition at 10° C. or lower. Because the hydrolysistreatment is performed at 10° C. or lower, generation or increase in atleast one impurity selected from the group consisting of conjugatedunsaturated fatty acids and coloring substances during the hydrolysisstep can be suppressed. The temperature condition of the hydrolysistreatment needs to be a temperature range that can proceed thehydrolysis treatment as long as the temperature condition is at 10° C.or lower. For example, the temperature condition can be at −20° C. orhigher, −10° C. or higher, −5° C. or higher, −4° C. or higher, −2° C. orhigher, 0° C. or higher, or 2° C. or higher and can be at 8° C. or loweror 7° C. or lower. The temperature range of the hydrolysis treatment maybe a numerical range of a combination of any upper limit value and anylower limit value described above. For example, the temperature rangemay be from −20° C. to 10° C., from −10° C. to 10° C., from −5° C. to10° C., from −4° C. to 10° C., from 0° C. to 10° C., from 0° C. to 8°C., or from 2° C. to 7° C. When the hydrolysis treatment is performed insuch a temperature condition at 10° C. or lower, generation or increasein the impurity described above can be further suppressed.

The reaction time of the alkali hydrolysis treatment differs dependingon the set temperature range and, for example, the reaction time may befrom 30 minutes to 600 hours, from 1 hour to 100 hours, from 8 hours to80 hours, or from 19 hours to 25 hours. The amount of the fatty acidalkyl ester in the reaction solution decreases as the alkali hydrolysistreatment proceeds. Therefore, the alkali hydrolysis treatment can beterminated depending on the amount of the fatty acid alkyl esterremaining in the reaction solution. The amount of the fatty acid alkylester in the reaction solution can be identified by thin-layerchromatography (TLC), high performance liquid chromatography (HPLC), orthe like.

The alkali hydrolysis treatment can be terminated by adding an acid tothe reaction solution. By the addition of the acid, the pH of thereaction solution becomes acidic. Thus, the progression of thehydrolysis reaction is terminated, and a saponified product produced bythe addition of the alkali catalyst is decomposed, thereby obtaining afree fatty acid. At this time, the free fatty acid, which is obtained bythe termination treatment of the reaction, can be extracted by allowingan organic solvent such as hexane to be present in the reactionsolution. Temperature conditions of the reaction termination and theextraction treatment are not particularly limited and, for example, thetemperature conditions may be in a range of 0° C. to 40° C., 5° C. to35° C., or 15° C. to 30° C. Timing of the reaction termination and theextraction treatment are not particularly limited and may be at the timewhen the reaction solution mixed by agitation or the like is separatedinto layers and stabilized.

The acid used for the termination of the alkali hydrolysis reaction ispublicly known in the art, and examples of the acid include inorganicacids, such as hydrochloric acid, sulfuric acid, phosphoric acid, nitricacid, and carbonic acid, or organic acids, such as acetic acid, citricacid, and oxalic acid. As the acid, an inorganic acid is preferable fromthe perspective of ease in removal by water washing due to its highsolubility to water. In particular, the acid is more preferablyhydrochloric acid or the like because the amount of addition needs to beonly a small amount and because generated salts and remaining acid canbe removed. The added amount of the acid needs to be an amount that iseffective in terminating the alkali hydrolysis treatment and may beapproximately 1.1 equivalents relative to the amount of the added alkalicatalyst.

The pH of the reaction solution after the acid addition needs to be a pHthat can terminate the alkali hydrolysis, and the lower limit valuethereof may be pH 1.0, pH 1.5, or pH 2.0 while the upper limit valuethereof may be pH 6.0, pH 5.0, pH 4.5, or pH 4.0. The pH of the reactionsolution after the acid addition may be, for example, from pH 1.0 to pH6.0, from pH 1.5 to pH 4.5, from pH 2.0 to pH 5.0, and from 2.0 to 4.0.When the pH of the reaction solution after the acid addition is set in arange that is greater than pH 1, for example, pH 1.5 to pH 4.5 or pH 2.0to pH 4.0, progression of a reverse reaction that may generate fattyacid alkyl esters after the termination of the hydrolysis reaction issuppressed, and increase in fatty acid alkyl esters can be suppressed.The pH of the reaction solution herein means a pH of an aqueous layer inthe reaction solution containing an organic layer and the aqueous layer.

This manufacturing method may include a washing step of removing awater-soluble component from the reaction solution obtained after thereaction termination and the extraction treatment. In the washing step,water or the like may be used as a wash liquid and added to the reactionsolution. The washing step may be performed until the pH of the washliquid used in the washing treatment reaches approximately neutral, forexample, greater than 6. The temperature of the washing step is notparticularly limited, and the washing step may be performed at 25° C. orlower. After the washing step, this manufacturing method may include arecovery step that recovers the target free LC-PUFA-containingcomposition from the organic layer of the reaction solution after thewashing treatment. The recovering treatment may employ techniquestypically used for this purpose and, for example, may use an evaporatoror the like.

The free LC-PUFA-containing composition obtained by this manufacturingmethod has a lower content of conjugated unsaturated fatty acids and/orcoloring substances than the content of the case where hydrolysistreatment is performed at a temperature of higher than 10° C. In thefree LC-PUFA-containing composition obtained by this manufacturingmethod, the content of the LC-PUFA may be, for example, 80.0% orgreater, 85.0% or greater, 90.0% or greater, 95% or greater, 97.0% orgreater, 98.0% or greater, 99.0% or greater, or 99.5% or greater, of thefatty acids in the composition. In the free LC-PUFA-containingcomposition obtained by this manufacturing method, the content ofconjugated unsaturated fatty acids is an amount that is lower than theamount of the case where hydrolysis treatment is performed at atemperature of higher than 10° C. and, for example, may be 1.0% or less,0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, or0.3% or less, of the fatty acids in the composition. In the freeLC-PUFA-containing composition obtained by this manufacturing method,the Gardner color as an indicator of coloring substances is a value thatis lower than the value of the case where hydrolysis treatment isperformed at a temperature of higher than 10° C. and, for example, maybe less than 3+, 3− or less, 2+ or less, 2 or less, 1+ or less, or 1−.Examples of such a free LC-PUFA-containing composition include freeLC-PUFA-containing compositions in other embodiments of the presentdisclosure described above.

The free LC-PUFA-containing composition has a smaller amount ofremaining enzyme that has undergone heat inactivation treatment comparedto the amount in a free LC-PUFA-containing composition obtained by usinga hydrolysis enzyme. Effect of the remaining enzyme can be reduced withthe composition having a smaller amount the remained heat-inactivatedenzyme.

The free LC-PUFA-containing composition can have a low amount ofresidual organic solvent because the free LC-PUFA-containing compositionis derived from a bio-oil and can be obtained without undergoing a stepof chemical synthesis. The organic solvent in the present specificationmeans an organic solvent other than fatty acids and means a hydrophobicor hydrophilic solvent having at least one carbon. Examples of theorganic solvent include polar solvents, nonpolar solvents,water-miscible solvents, water-immiscible solvents, and combinations orat least two of these. Examples of the organic solvent includesubstituted or unsubstituted, saturated or unsaturated aliphatichydrocarbons, aromatic hydrocarbons, alcohols, ethers, ketones,aldehydes, carboxylic acids, esters, nitriles, amides and the like. Theorganic solvent may be one type of these or a combination of at leasttwo of these.

The total content of the residual organic solvent in the freeLC-PUFA-containing composition may be 5000 ppm or less, 3000 ppm orless, 2000 ppm or less, or 1000 ppm or less.

The free LC-PUFA-containing composition may have a low content of atleast one selected from the group consisting of methanol, ethanol,acetone, and hexane among the residual organic solvents. The content ofthese organic solvent may be each independently 500 ppm or less, 300 ppmor less, or 200 ppm or less. For example, all of the contents ofmethanol, ethanol, acetone, and hexane in the free LC-PUFA-containingcomposition may be 500 ppm or less, 300 ppm or less, or 200 ppm or less.

Because the free LC-PUFA-containing composition contains a smalleramount of impurities described above and contains a high concentrationof at least one free LC-PUFA, functions corresponding to the type of theLC-PUFA contained can be favorably exhibited at high levels, and thefree LC-PUFA-containing composition can be preferably used for variouspurposes.

Examples of preferable applications of the free LC-PUFA-containingcomposition include usage in food products, supplements, medicaments,cosmetics, and animal feed and usage in the manufacturing methodstherefor. In particular, the free LC-PUFA-containing composition may bepreferably used in medicaments containing a composition containing theLC-PUFA as an active ingredient. For example, when this freeLC-PUFA-containing composition is a composition containing free ARA,free DGLA, free EPA, free DHA, or the like, the free LC-PUFA-containingcomposition can be significantly advantageously applied for the purposesrequiring high productivity and high content of these functionalLC-PUFA. Examples of such purposes include food products, supplements,medicaments, cosmetics, animal feed, and the like that are expected toexhibit effect on prevention of lifestyle-related diseases, such asarteriosclerosis, cerebral infarction, myocardial infarction,thrombosis, and hyperlipemia, improvement of metabolic syndrome,antiallergy, antiinflammation, anticancer, improvement in brainfunctions. Examples of the medicament include external medicines forskin, oral preparations and the like.

When the free LC-PUFA-containing composition is used as a medicament,the medicament contains the free LC-PUFA-containing composition and apharmaceutically acceptable carrier and, as necessary, other components.The dosage form may be any form that is convenient for oraladministration or parenteral administration based on the type of theLC-PUFA in the composition. Examples of the dosage form includeinjections, transfusions, powders, granules, tablets, capsules, entericcoated tablets, troches, peroral liquid preparations, suspensions,emulsions, syrups, liquids for external use, fomentations, nasalpreparations, eardrops, eye drops, inhalants, ointments, lotions,suppositories and the like. These may be used individually or incombination depending on the symptoms.

By normal methods, these various types of preparations, according to apurpose, may be formulated by adding, to the principle agent, previouslyknown adjutants commonly used in the field of drug preparationtechnology, as exemplified by excipients, binders, preservatives,stabilizers, disintegrants, lubricants, flavoring agents, or the like.Furthermore, in the case of oral administration for an adult, typically,the dosage for administration can be appropriately adjusted in a rangeof 0.01 mg to 10 g, preferably 0.1 mg to 2 g, and more preferably 1 mgto 200 mg, per day as the total amount of the LC-PUFA as a structuredlipid. In the case of parenteral administration, the dosage foradministration can be appropriately adjusted in a range of 0.001 mg to 1g, preferably 0.01 mg to 200 mg, and more preferably 0.1 mg to 100 mg,per day as the total amount of the LC-PUFA as a structured lipid.However, these dosages differ depending on purpose of theadministration, type of the LC-PUFA in the composition, and conditionsof the person subjected to the administration (sex, age, weight, and thelike).

EXAMPLES

The present disclosure is described below in detail using examples.However, the present disclosure is not limited in any manner by theseexamples.

In the examples and comparative examples in the section below, theLC-PUFA refers only to particular types; however, the type of theLC-PUFA is not particularly limited.

“Purified water” used in the section of examples below means water thathas been purified, and “water” means tap water.

It was supposed that most of the fatty acids contained in the fatty acidalkyl ester-containing composition used in the examples as a rawmaterial composition is in a fatty acid alkyl ester form. Consequently,the fatty acids contained in the samples are all described below asfatty acids in the alkyl ester form. However, this does not completelynegate the fact that fatty acids in a form other than an alkyl esterform are included.

Comparative Example 1

Preparation Method

A raw material EPA ethyl ester 1 derived from a fish oil containing96.7% of EPA was alkali-hydrolyzed by a conventional method.

That is, to 2.50 g of the raw material EPA ethyl ester 1, 6.25 mL ofethanol (4.92 g, 14.11 equivalents relative to the amount of fattyacids), 1.00 mL of water, and 0.76 g of 48 wt. % sodium hydroxideaqueous solution (1.20 equivalents of base relative to the amount offatty acids) were added to prepare a sample solution 1. In the samplesolution 1, the water content was 1.40 g. that is, 10.27 equivalentsrelative to the amount of fatty acids. The sample solution 1 was heatedby an oil bath at 70° C. for 24 hours to perform hydrolysis treatment.In Table 1, the composition used during the preparation of the samplesolution 1 is shown. In Table 1, the raw material composition indicatesthe weight (g) in the sample solution, and amounts of base, ethanol, andwater indicates amounts relative to the amount of fatty acids in the rawmaterial composition (molar equivalent). Note that, in the samplesolution after the hydrolysis reaction, the amount of ethanol relativeto the amount of fatty acids may be, theoretically, increased by at most1 equivalent. In the sample solution after the hydrolysis reaction, theamount of water relative to the amount of fatty acids may be,theoretically, decreased by at most 1 equivalent. This is the samehereafter.

The termination of the hydrolysis treatment reaction was determined asfollows. That is, a part of the sample solution 1 was taken out andcombined and mixed in the ratio, sample solution: 1N hydrochloric acidaqueous solution:hexane=1:2:5 (v/v/v). The separated hexane layer wasused as a sample for identification.

Onto a TLC plate, 0.5 μL of the sample for identification was loaded byusing a microsyringe and developed in a developing chamber. After thedevelopment, the thin-layer plate was taken out from the developingchamber, the solvent was vaporized in a fume hood, and a p-anisaldehydecoloring reagent was applied by dipping. After the application, heatingwas performed at approximately from 110° C. to 120° C. until colordeveloped, thereby obtaining a spot. Disappearance of the spot of theraw material ethyl ester was visually observed and used as the point ofreaction termination. This is the same hereafter.

As the development solvent, a solvent in whichhexane:diethylether:acetic acid was 80:20:1 (v/v/v) was used. As the TLCplate, Silica gel 60G F254 (Merck Millipore) was used. As the coloringagent, a p-anisaldehyde coloring reagent was used.

The p-anisaldehyde coloring reagent was prepared as described below.That is, after 9.3 mL of p-anisaldehyde, 3.8 mL of acetic acid, and 340mL of ethanol were mixed while being cooled with ice, 12.5 mL ofconcentrated sulfuric acid was mixed to the mixture to prepare thep-anisaldehyde coloring reagent.

The sample solution 1 after the treatment was air-cooled and transferredinto a separatory funnel, and then 3.13 mL of hexane and 2.50 mL ofpurified water were added to this sample solution 1. After 2.25 g ofhydrochloric acid was further added to the sample solution 1, the samplesolution 1 was separated into two layers, a hexane layer and an aqueouslayer. The pH of the aqueous layer was 1.0. In Table 1, the pH of theaqueous layer at this time was referred to as “pH at acidification”.This is the same hereafter.

The sample solution 1 was agitated and then allowed to stand still.Then, after the aqueous layer was removed from the sample solution 1,3.75 mL of purified water was further added to the sample solution 1 andagitated. An extremely small amount of hydrochloric acid was added toadjust the pH of the aqueous layer to 1.0. Thereafter, water washing wasperformed by using the same amount of purified water as a liquid forwater washing. Water washing was repeated until the liquid for waterwashing collected after the water washing became neutral, pH 6.0 to 7.0.The hexane layer was recovered from the sample solution 1 after thewater washing. From the recovered hexane layer, hexane was removed by anevaporator and vacuum drawing, and 2.12 g of EPA 1, which was acomposition containing free EPA, was obtained.

Evaluation Method

The following evaluations were performed for the raw material EPA ethylester 1 and the EPA 1 obtained as described above. Among the evaluationresults, Table 1 shows characteristics of the EPA 1, and Table 4 showsthe fatty acid composition.

The recovery percentage was 96.8%. The Gardner color of the obtained EPA1 was 6−, AnV was 1.9, the ethyl ester (EE) content was 2820 ppm, andthe conjugated dienoic acid content was 2.45%. Conjugated unsaturatedfatty acids other than the conjugated dienoic acid were not detected.Note that, for the conjugated unsaturated fatty acid, only theconjugated dienoic acid is shown in Table 1.

(1) Recovery Percentage

The recovery percentage of EPA of the EPA 1 was determined from thefollowing equation. The molecular weight of the EPA ethyl ester as theraw material composition was 330.5028, and the molecular weight of thefree EPA as the hydrolysate was 302.4498.

$\begin{matrix}{{{recovery}\mspace{14mu}{percentage}\mspace{14mu}(\%)} = {\frac{{weight}\mspace{14mu}{of}\mspace{14mu}{hydrolysate}\mspace{14mu}(g)}{{molecular}\mspace{14mu}{weight}\mspace{14mu}{of}\mspace{14mu}{hydrolysate}\mspace{14mu}(g)} \times \frac{{molecular}\mspace{14mu}{weight}\mspace{14mu}{of}\mspace{14mu}{raw}\mspace{14mu}{material}\mspace{14mu}{composition}}{{weight}\mspace{14mu}{of}\mspace{14mu}{raw}\mspace{14mu}{material}\mspace{14mu}{composition}\mspace{14mu}(g)} \times 100}} & \left\lbrack {{Equation}\mspace{14mu} 1} \right\rbrack\end{matrix}$(2) Conjugated Unsaturated Fatty Acid

The measurement was performed based on Reference 1.14 of the StandardMethods for the Analysis of Fats, Oils, and Related Materials, 2013Edition, established by Japan Oil Chemists' Society (JOCS).

(3) Color

The Gardner color was determined based on 3.2.1.1 of the StandardMethods for the Analysis of Fats, Oils, and Related Materials, 2013Edition, established by Japan Oil Chemists' Society (JOCS).

(4) Anisidine Value

The anisidine value was determined based on 2.5.3 of the StandardMethods for the Analysis of Fats, Oils, and Related Materials, 2013Edition, established by Japan Oil Chemists' Society (JOCS).

(5) Fatty Acid Composition

Fatty acid compositions of the raw material EPA ethyl ester 1 and theEPA 1 were determined from each fatty acid peak obtained by gaschromatography performed in the condition described below. Note that,for the EPA 1, methyl esterification was performed before the gaschromatography analysis. The methyl esterification was performed inaccordance with American Oil Chemists' Society (AOCS) Official Method Ce1b-89. Gas chromatography analysis conditions

Instrument: Agilent 7890 GC system (Agilent Technologies)

Column: DB-WAX (Agilent Technologies, 30 m×0.25 mm ID, 0.25 μm filmthickness) J&W122−7032

Column oven: 180° C.-3° C./min-230° C. (25 min)

Injection temperature: 250° C.

Injection method: split

Split ratio: 30:1

Detector temperature: 270° C.

Detector: FID

Carrier gas: helium (1.0 mL/min, constant flow)

(6) Fatty Acid Ethyl Ester Content

The measurement was performed by high performance liquid chromatography(HPLC) in the following conditions. A sample solution was prepared bydiluting the EPA 1 with a 5 v/v % acetic acid/acetone solution under acondition that the concentration of the EPA 1 became 10 mg/mL. For thequantification, the measurement was performed by using a productcontaining 99% EPA ethyl ester and appropriately creating a calibrationcurve. HPLC measurement conditions

Column: YMC_ODS-A column (4.6 mm×150 mm)

Eluent: MeOH:water=9:1

Flow rate: 1 mL/min

Column temperature: 40° C.

Injection amount: 5 μL was injected

Detector: UV 205 nm

Comparative Example 2

Alkali hydrolysis was performed in the same manner as in ComparativeExample 1 except for changing the amount of the raw material EPA ethylester 1 containing 96.7% of EPA of Comparative Example 1 and the amountof the ethanol in the sample solution to the amounts described below.

That is, to 5.00 g of the raw material EPA ethyl ester 1, 3.50 mL ofethanol (2.75 g, 3.95 equivalents relative to the amount of fattyacids), 2.01 mL of water, and 1.51 g of 48 wt. % sodium hydroxideaqueous solution (1.20 equivalents of base relative to the amount offatty acids) were added to prepare a sample solution 2. In the samplesolution 2, the water content was 2.80 g, that is, 10.27 equivalentsrelative to the amount of fatty acids. The sample solution 2 was heatedwith an oil bath at 70° C. for 24 hours to perform hydrolysis treatment.After the confirmation of termination of the hydrolysis treatmentreaction, the sample solution 2 after the treatment was air-cooled andtransferred into a separatory funnel, and then 6.25 mL of hexane and5.00 mL of purified water were added to this sample solution 2. After2.02 g of hydrochloric acid was further added to the sample solution 2,the sample solution 2 was separated into two layers, a hexane layer andan aqueous layer. The pH of the aqueous layer was 3.0.

The sample solution 2 was agitated and then allowed to stand still.Then, after the aqueous layer was removed from the sample solution 2,7.50 mL of purified water was further added to the sample solution 2after the removal and agitated. An extremely small amount ofhydrochloric acid was added to adjust the pH of the aqueous layer to1.0. Thereafter, water washing was performed by using the same amount ofpurified water as a liquid for water washing. Water washing was repeateduntil the liquid for water washing collected after the water washingbecame neutral, pH 6.0 to 7.0. The hexane layer was recovered from thesample solution 2 after the water washing. From the recovered hexanelayer, hexane was removed by an evaporator and vacuum drawing, and 4.17g of EPA 2, which was a composition containing free EPA, was obtained.

For the raw material EPA ethyl ester 1 and the EPA 2 obtained asdescribed above, the evaluations were performed in the same manner asfor the EPA 1. The results are shown in Table 1 and Table 4.

The recovery percentage was 91.0%. The Gardner color of the obtained EPA2 was 12, AnV was 5.3, the ethyl ester (EE) content was 191 ppm, and theconjugated dienoic acid amount was 1.12%. Conjugated unsaturated fattyacids other than the conjugated dienoic acid were not detected. Notethat, for the conjugated unsaturated fatty acid, only the conjugateddienoic acid is shown in Table 1.

Example 1

Alkali hydrolysis was performed in the same manner as in ComparativeExample 1 except for changing the temperature condition during thealkali hydrolysis of the raw material EPA ethyl ester 1 containing 96.7%of EPA of Comparative Example 1 to 6° C.

That is, to 2.50 g of the raw material EPA ethyl ester 1, 6.25 mL ofethanol (4.92 g, 14.11 equivalents relative to the amount of fattyacids), 1.00 mL of water, and 0.76 g of 48 wt. % sodium hydroxideaqueous solution (1.20 equivalents of base relative to the amount offatty acids) were added to prepare a sample solution 3. In the samplesolution 3, the water content was 1.40 g, that is, 10.27 equivalentsrelative to the amount of fatty acids. The sample solution 3 wasagitated at 6° C. for 24 hours to perform hydrolysis treatment. Afterthe confirmation of termination of the hydrolysis treatment reaction,the sample solution 3 after the treatment was returned to roomtemperature and transferred into a separatory funnel, and then 3.13 mLof hexane and 2.50 mL of purified water were added to this samplesolution 3. After 2.25 g of hydrochloric acid was further added to thesample solution 3, the sample solution 3 was separated into two layers,a hexane layer and an aqueous layer. The pH of the aqueous layer was1.0.

The sample solution 3 was agitated and then allowed to stand still.Then, after the aqueous layer was removed from the sample solution 3,3.75 mL of purified water was further added in the sample solution 3after the removal and agitated. An extremely small amount ofhydrochloric acid was added to adjust the pH of the aqueous layer to1.0. Thereafter, water washing was performed by using the same amount ofpurified water as a liquid for water washing. Water washing was repeateduntil the liquid for water washing collected after the water washingbecame neutral, pH 6.0 to 7.0. The hexane layer was recovered from thesample solution 3 after the water washing. From the recovered hexanelayer, hexane was removed by an evaporator and vacuum drawing, and 2.14g of EPA 3, which was a composition containing free EPA, was obtained.

For the EPA 3, the evaluations were performed in the same manner as forthe EPA 1. The results are shown in Table 1 and Table 4.

The recovery percentage was 93.8%. The Gardner color of the obtained EPA3 was 2−. AnV was 1.3, the ethyl ester (EE) content was 2790 ppm, andthe conjugated dienoic acid content was 0.47%. Conjugated unsaturatedfatty acids except the conjugated dienoic acid were not detected. Thesephysical property values are shown in Table 1. Note that, for theconjugated unsaturated fatty acid, only the conjugated dienoic acid isshown in Table 1.

Example 2

Alkali hydrolysis was performed in the same manner as in ComparativeExample 2 except for changing the temperature condition during thealkali hydrolysis of the raw material EPA ethyl ester 1 containing 96.7%of EPA of Comparative Example 1 to 6° C.

That is, to 5.00 g of the raw material EPA ethyl ester 1, 3.50 mL ofethanol (2.75 g, 3.95 equivalents relative to the amount of fattyacids), 2.01 mL of water, and 1.51 g of 48 wt. % sodium hydroxideaqueous solution (1.20 equivalents of base relative to the amount offatty acids) were added to prepare a sample solution 4. In the samplesolution 4, the water content was 2.80 g, that is, 10.27 equivalentsrelative to the amount of fatty acids. The sample solution 4 wasagitated at 6° C. for 24 hours to perform hydrolysis treatment.

The sample solution 4 after the treatment was returned to roomtemperature and transferred into a separatory funnel, and then 6.25 mLof hexane and 5.00 mL of purified water were added to this samplesolution 4. After 2.09 g of hydrochloric acid was further added to thesample solution 4, the sample solution 4 was separated into two layers,a hexane layer and an aqueous layer. The pH of the aqueous layer was3.0.

The sample solution 4 was agitated and then allowed to stand still.Then, after the aqueous layer was removed from the sample solution 4,7.50 mL of purified water was further added in the sample solution 4after the removal and agitated. An extremely small amount ofhydrochloric acid was added to adjust the pH of the aqueous layer to1.0. Thereafter, water washing was performed by using the same amount ofpurified water as a liquid for water washing. Water washing was repeateduntil the liquid for water washing collected after the water washingbecame neutral, pH 6.0 to 7.0. The hexane layer was recovered from thesample solution 4 after the water washing. From the recovered hexanelayer, hexane was removed by an evaporator and vacuum drawing, and 4.37g of EPA 4, which was a composition containing free EPA, was obtained.

For the EPA 4, the evaluations were performed in the same manner as forthe EPA 1. The results are shown in Table 1 and Table 4.

The recovery percentage was 95.4%. The Gardner color of the obtained EPA4 was 1, AnV was 2.1, the ethyl ester (EE) content was 240 ppm, and theconjugated dienoic acid content was 0.39%. Conjugated unsaturated fattyacids except the conjugated dienoic acid were not detected. Note that,for the conjugated unsaturated fatty acid, only the conjugated dienoicacid is shown in Table 1.

Comparative Example 3

Alkali hydrolysis was performed in the same manner as in ComparativeExample 2 except for using a raw material DGLA ethyl ester 1 that wasderived from microorganisms and that contained 96.1% of DGLA.

That is, to 1.50 g of the raw material DGLA ethyl ester 1, 1.05 mL ofethanol (0.83 g, 4.00 equivalents relative to the amount of fattyacids), 0.61 mL of water, and 0.45 g of 48 wt. % sodium hydroxideaqueous solution (1.20 equivalents of base relative to the amount offatty acids) were added to prepare a sample solution 5. In the samplesolution 5, the water content was 0.84 g, that is, 10.40 equivalentsrelative to the amount of fatty acids. The sample solution 5 was heatedby an oil bath at 70° C. for 24 hours to perform hydrolysis treatment.

After the confirmation of termination of the hydrolysis treatmentreaction, the sample solution 5 after the treatment was air-cooled andtransferred into a separatory funnel, and then 1.88 mL of hexane and1.50 mL of purified water were added to this sample solution 5. After0.60 g of hydrochloric acid was further added to the sample solution 5,the sample solution 5 was separated into two layers, a hexane layer andan aqueous layer. The pH of the aqueous layer was 3.0.

The sample solution 5 was agitated and then allowed to stand still.Then, after the aqueous layer was removed from the sample solution 5,2.25 mL of purified water was further added in the sample solution 5after the removal and agitated. An extremely small amount ofhydrochloric acid was added to adjust the pH of the aqueous layer to1.0. Thereafter, water washing was performed by using the same amount ofpurified water as a liquid for water washing. Water washing was repeateduntil the liquid for water washing collected after the water washingbecame neutral, pH 6.0 to 7.0. The hexane layer was recovered from thesample solution 5 after the water washing. From the recovered hexanelayer, hexane was removed by an evaporator and vacuum drawing, and 1.19g of DGLA 1, which was a composition containing free DGLA, was obtained.

For the raw material DGLA ethyl ester 1 and the DGLA 1 obtained asdescribed above, the evaluations were performed in the same manner asfor Comparative Example 2. The results are shown in Table 2 and Table 4.The recovery percentage was 86.4%. The Gardner color of the obtainedDGLA 1 was 6−, AnV was 13.7, the ethyl ester (EE) content was 21 ppm,and the conjugated dienoic acid content was 1.03%. Conjugatedunsaturated fatty acids except the conjugated dienoic acid were notdetected. Note that, for the conjugated unsaturated fatty acid, only theconjugated dienoic acid is shown in Table 2.

Note that, to measure the fatty acid ethyl ester content, a samplesolution was prepared by diluting the DGLA 1 with a 5 v/v % aceticacid/acetone solution under a condition that the concentration of theDGLA 1 became 10 mg/mL, and a calibration curve was created by using aproduct containing 99% DGLA ethyl ester. For the calculation of therecovery percentage, the molecular weight of the free DGLA as ahydrolysate was 306.48, and the molecular weight of the DGLA ethyl esteras a raw material composition was 334.53.

Example 3

Alkali hydrolysis was performed in the same manner as in ComparativeExample 3 except for using a raw material DGLA ethyl ester 2 that wasderived from microorganisms and that contained 96.1% of DGLA, andchanging the temperature condition during the hydrolysis to 6° C. As aresult, 1.31 g of DGLA 2, which was a composition containing free DGLA,was obtained.

For the raw material DGLA ethyl ester 2 and the DGLA 2 obtained asdescribed above, the evaluations were performed in the same manner asfor Comparative Example 3. The results are shown in Table 2 and Table 4.The recovery percentage was 95.2%. The Gardner color of the obtainedDGLA 2 was 1−, AnV was 3.2, the ethyl ester (EE) content was 151 ppm,and the conjugated dienoic acid content was 0.24%. Conjugatedunsaturated fatty acids except the conjugated dienoic acid were notdetected. Note that, for the conjugated unsaturated fatty acid, only theconjugated dienoic acid is shown in Table 2.

Comparative Example 4

Alkali hydrolysis was performed in the same manner as in ComparativeExample 1 except for using a raw material PUFA ethyl ester 1 that wasderived from fish oil and that contained 39.0% of DHA and 47.8% of EPA.

That is, to 2.50 g of the raw material PUFA ethyl ester 1, 6.25 mL ofethanol (4.92 g, 14.53 equivalents relative to the amount of fattyacids), 1.02 mL of water, and 0.73 g of 48 wt. % sodium hydroxideaqueous solution (1.20 equivalents of base relative to the amount offatty acids) were added to prepare a sample solution 7. In the samplesolution 7, the water content was 1.40 g, that is, 10.57 equivalentsrelative to the amount of fatty acids. The sample solution 7 was heatedwith an oil bath at 70° C. for 24 hours to perform hydrolysis treatment.

The sample solution 7 after the treatment was air-cooled and transferredinto a separatory funnel, and then 3.13 mL of hexane and 2.50 mL ofpurified water were added to this sample solution 7. Then, hydrochloricacid was further added to the mixture. The sample solution 7 wasseparated into two layers, a hexane layer and an aqueous layer, afterthe addition of the hydrochloric acid. The pH of the aqueous layer was1.0.

The sample solution 7 was agitated and then allowed to stand still.Then, after the aqueous layer was removed from the sample solution 7,3.75 mL of purified water was further added to the sample solution 7after the removal and agitated. An extremely small amount ofhydrochloric acid was added to adjust the pH of the aqueous layer to1.0. Thereafter, water washing was performed by using the same amount ofpurified water as a liquid for water washing. Water washing was repeateduntil the liquid for water washing collected after the water washingbecame neutral, pH 6.0 to 7.0. The hexane layer was recovered from thesample solution 7 after the water washing. From the recovered hexanelayer, hexane was removed by an evaporator and vacuum drawing, and 2.25g of PUFA 1, which was a composition containing free DHA and free EPA,was obtained.

For the raw material PUFA ethyl ester 1 and the PUFA 1 obtained asdescribed above, the evaluations were performed in the same manner asfor Comparative Example 1. The results are shown in Table 3 and Table 4.The recovery percentage was 98.4%. The Gardner color of the obtainedDGLA 1 was 7−, AnV was 2.1, and the conjugated dienoic acid content was3.29%. Conjugated unsaturated fatty acids except the conjugated dienoicacid were not detected. Note that, for the conjugated unsaturated fattyacid, only the conjugated dienoic acid is shown in Table 3.

Note that, for the calculation of the recovery percentage, the averagemolecular weight of the fatty acids in the raw material PUFA ethyl ester1 was 340.167, and the average molecular weight of the fatty acids inthe obtained PUFA 1 was 312.167.

Example 4

Alkali hydrolysis was performed in the same manner as in ComparativeExample 4 except for using a raw material PUFA ethyl ester 2 that wasderived from fish oil and that contained 39.0% of DHA and 47.8% of EPA,and changing the temperature condition during the alkali hydrolysis to6° C. As a result, 4.58 g of PUFA 2, which was a composition containingfree PUFA, was obtained.

That is, to 5.00 g of the raw material PUFA ethyl ester 2, 3.50 mL ofethanol (2.75 g, 4.07 equivalents relative to the amount of fattyacids), 2.04 mL of water, and 1.47 g of 48 wt. % sodium hydroxideaqueous solution (1.20 equivalents of base relative to the amount offatty acids) were added to prepare a sample solution 8. In the samplesolution 8, the water content was 2.80 g, that is, 10.57 equivalentsrelative to the amount of fatty acids. The sample solution 8 wasagitated at 6° C. for 24 hours to perform hydrolysis treatment.

After the confirmation of termination of the hydrolysis treatmentreaction, the sample solution 8 after the treatment was air-cooled andtransferred into a separatory funnel, and then 6.25 mL of hexane and5.00 mL of purified water were added to this sample solution 8. Then,hydrochloric acid was further added to the mixture. The sample solution8 was separated into two layers, a hexane layer and an aqueous layer.The pH of the aqueous layer was 3.0.

The sample solution 8 was agitated and then allowed to stand still.Then, after the aqueous layer was removed from the sample solution 8,7.50 mL of purified water was further added in the sample solution 8after the removal and agitated. An extremely small amount ofhydrochloric acid was added to adjust the pH of the aqueous layer to1.0. Thereafter, water washing was performed by using the same amount ofpurified water as a liquid for water washing. Water washing was repeateduntil the liquid for water washing collected after the water washingbecame neutral, pH 6.0 to 7.0. The hexane layer was recovered from thesample solution 8 after the water washing. From the recovered hexanelayer, hexane was removed by an evaporator and vacuum drawing, and 4.58g of PUFA 2, which was a composition containing free PUFA, was obtained.

For the raw material PUFA ethyl ester 2 and the PUFA 2 obtained asdescribed above, the evaluations were performed in the same manner asfor Comparative Example 4. The results are shown in Table 3 and Table 4.The recovery percentage was 99.7%. The Gardner color of the obtainedPUFA 2 was 2+, AnV was 3.9, and the conjugated dienoic acid content was0.65%. Conjugated unsaturated fatty acids except the conjugated dienoicacid were not detected. Note that, for the conjugated unsaturated fattyacid, only the conjugated dienoic acid is shown in Table 3.

Table 1 to Table 4 for Comparative Example 1 to Comparative Example 4and Example 1 to Example 4 are shown below. In Table 1 to Table 3,“Conditions” means the conditions of the hydrolysis treatment, and“Characteristics” means the characteristics and physical properties ofthe obtained composition by the hydrolysis treatment. In the columns ofcomparative examples and examples of Table 1 to Table 3, the left sidecolumns describe about the raw material compositions, and the right sidecolumns describe about the compositions obtained by the hydrolysistreatment. In Table 1 to Table 3, “EE” is an abbreviation for ethylester, and “G color” indicates the Gardner color. In Table 4, “C18PUFA”means the total of the di- or higher-valent polyunsaturated fatty acidhaving 18 carbons. “Others” means substances that are fatty acids exceptthe LC-PUFA and the C18PUFA and that are not written. The “Others” is avalue calculated by subtracting the content of the LC-PUFA and thecontent of the C18PUFA from the numerical value of 100%. “n.d.” meansless than 0.01%.

TABLE 1 Comparative Comparative Example 1 Example 2 Example 1 Example 2EPA- EPA- EPA- EPA- EE 1 EPA 1 EE 1 EPA 2 EE 1 EPA 3 EE 1 EPA 4Conditions Temperature — 70° C. — 70° C. — 6° C. — 6° C. Sample Rawmaterial — 2.5 g — 5.0 g — 2.5 g — 5.0 g solution compositioncomposition Base — 1.20 — 1.20 — 1.20 — 1.20 equivalents equivalentsequivalents equivalents Ethanol — 14.11 — 3.95 — 14.11 — 3.95equivalents equivalents equivalents equivalents Water — 10.27 — 10.27 —10.27 — 10.27 equivalents equivalents equivalents equivalents pH atacidification — 1   — 3 — 1   — 3 Characteristics LC-PUFA (%) 97.24%96.15% 96.78% 97.07% 97.01% G color 1−  6−  1− 12 1− 2−  1− 1 AnV 1.41.9 1.2 5.3 1.2 1.3 1.2 2.1 EE content (ppm) — 2820    — 191 — 2790    —240 Conjugated dienoic acid (%) 0.40  2.45 0.40 1.12 0.40  0.47 0.400.39 Recovery percentage (%) — 96.8  — 91.0 — 93.8  — 95.4

TABLE 2 Comparative Example 3 Example 3 DGLA- DGLA- EE 1 DGLA 1 EE 2DGLA 2 Conditions Temperature — 70° C. — 6° C. Sample Raw material — 1.5g — 1.5 g solution composition composition Base — 1.20 — 1.20equivalents equivalents Ethanol — 4.00 — 4.00 equivalents equivalentsWater — 10.40 — 10.40 equivalents equivalents pH at acidification — 3  —3 Characteristics LC-PUFA (%) 97.47% 97.02% 97.22% G color 1−  6− 1−  1−AnV 2.2 13.7 1.5   3.2 EE content (ppm) — 21   — 151  Conjugated dienoicacid (%) 0.22  1.03 0.22   0.24 Recovery percentage (%) — 86.4 —  95.2

TABLE 3 Comparative Example 4 Example 4 PUFA- PUFA- EE 1 PUFA 1 EE 2PUFA 2 Conditions Temperature — 70° C. — 6° C. Sample Raw material — 2.5g — 5.0 g solution composition composition Base — 1.20 — 1.20equivalents equivalents Ethanol — 14.53 — 4.07 equivalents equivalentsWater — 10.57 — 10.57 equivalents equivalents pH at acidification — 1 —3 Characteristics LC-PUFA (%) 90.19% 89.36% 90.20% G color 1−  7− 1−  2+AnV 2.7   2.1 2.5   3.9 Conjugated dienoic acid (%) 0.63   3.29 0.63  0.65 Recovery percentage (%) —  98.4 —  99.7

TABLE 4 Comparative Comparative Comparative Comparative EPA- Example 1Example 2 Example 1 Example 2 DGLA- Example 3 Example 3 Example 4Example 4 EE 1 EPA 1 EPA 2 EPA 3 EPA 4 EE 1 DGLA 1 DGLA 2 PUFA-EE 1 PUFA1 PUFA 2 C20:2 0.02% n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.C20:3n-6 n.d. n.d. n.d. n.d. n.d. 96.14% 95.56% 95.76% n.d. n.d. n.d.C20:4n-6 0.13% 0.11% 0.11% 0.12% 0.13% 1.24% 1.38% 1.38% 0.06% 0.07%0.06% C20:3n-3 n.d. n.d. n.d. n.d. n.d. 0.09% 0.08% 0.08% n.d. n.d. n.d.C20:4n-3 0.32% 0.31% 0.31% 0.31% 0.31% n.d. n.d. n.d. 0.16% 0.16% 0.16%C20:5n-3 96.73% 95.73% 96.36% 96.64% 96.57% n.d. n.d. n.d. 47.77% 47.29%47.40% C21:5n-3 0.04% n.d. n.d. n.d. n.d. n.d. n.d. n.d. 0.35% 0.36%0.34% C22:5n-3 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 2.86% 2.82% 2.78%C22:6n-3 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 38.99% 38.66% 39.46%PUFA 97.24% 96.15% 96.78% 97.07% 97.01% 97.47% 97.02% 97.22% 90.19%89.36% 90.20% C18PUFA 0.10% 0.05% 0.05% 0.05% 0.05% 0.82% 0.80% 0.80%0.20% 0.15% 0.13% Others 2.66% 3.80% 3.17% 2.88% 2.94% 1.71% 2.18% 1.98%9.61% 10.49% 9.67%

As shown in Table 1 to Table 3, all of the compositions of Examples 1 to4, in which the alkali hydrolysis treatment was performed at 6° C., i.e.the EPA 3 and the EPA 4 containing free EPA, the DGLA 2 containing freeDGLA, and the PUFA 2 containing free DHA and free EPA, had conjugateddienoic acid contents of 1.0% or less and the Gardner colors of lessthan 3+. In particular, it was confirmed that the conjugated dienoicacid underwent almost no change even when subjected to silica geltreatment. Meanwhile, in all of Examples 1 to 4, the contents of theconjugated dienoic acid were reduced to at most the half of the contentsin the cases of Comparative Examples 1 to 4, in which the treatment wasperformed at 70° C.

Furthermore, in all of the compositions of Examples 1 to 3, theanisidine values were 5.0 or less, and the ethyl ester contents were 500ppm (0.05 wt. %) or less. In all of the compositions of Example 1 toExample 4, the amount of methanol, the amount of ethanol, the amount ofacetone, and the amount of hexane were all 200 ppm or less.

Between Example 1 and Example 2, the amount of the ethanol relative tothe amount of the raw material composition in the sample solution andthe pH at acidification were different. Compared to the EPA 3 containingthe free EPA obtained in Example 1, the EPA 4 containing the free EPAobtained in Example 2 had even better Gardner color, even lower amountof the fatty acid ethyl ester, and even lower content of the conjugateddienoic acid.

In such a free LC-PUFA-containing composition, amount of impuritiesincluding conjugated dienoic acid and/or coloring substance was small,and the high content of the target LC-PUFA was achieved. Such a freeLC-PUFA-containing composition can be favorably applied to the purposes,such as medical purpose.

Disclosure of JP 2015−170856 A filed on Aug. 31, 2015 is incorporatedherein in its entirety by reference.

All documents, patent applications, and technical specifications statedin the present specification are incorporated by citation in the presentspecification to the same degree as if stated to be incorporated byreference specifically and individually.

What is claimed is:
 1. A free polyunsaturated fatty acid-containingcomposition, comprising at least one free polyunsaturated fatty acidhaving 20 or more carbons in an amount that a content thereof is 80.0%or greater of fatty acids in the composition, wherein the freepolyunsaturated fatty acid-containing composition satisfies at least oneselected from the group consisting of conditions (1) and (2): (1) acontent of a conjugated unsaturated fatty acid being from 0.001% to 1.0%of the fatty acids in the composition; and (2) the Gardner color beingless than 3+.
 2. The free polyunsaturated fatty acid-containingcomposition according to claim 1, wherein the free polyunsaturated fattyacid-containing composition has an anisidine value of 5.0 or less. 3.The free polyunsaturated fatty acid-containing composition according toclaim 1, wherein a content of the fatty acid alkyl ester is 0.2% or lessof the fatty acids in the composition.
 4. The free polyunsaturated fattyacid-containing composition according to claim 1, wherein the content ofthe conjugated unsaturated fatty acid is from 0.01% to 0.8% of the fattyacids in the composition.
 5. The free polyunsaturated fattyacid-containing composition according to claim 1, wherein thepolyunsaturated fatty acid is at least one selected from the groupconsisting of eicosadienoic acid, dihomo-y-linolenic acid, Mead acid,eicosatetraenoic acid, arachidonic acid, eicosapentaenoic acid,docosatetraenoic acid, docosapentaenoic acid, and docosahexaenoic acid.6. The free polyunsaturated fatty acid-containing composition accordingto claim 1, wherein a total content of a residual organic solvent in thecomposition is 5000 ppm or less.
 7. The free polyunsaturated fattyacid-containing composition according to claim 1, wherein a content of adi- or higher-valent polyunsaturated fatty acid having 18 carbons in thecomposition is 2.0% or less of the fatty acids in the composition.